The bioelectrical properties of pancreatic islet cells: Effect of diabetogenic agents

Diabetologia, Jun 1972

P. M. Dean, E. K. Matthews

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The bioelectrical properties of pancreatic islet cells: Effect of diabetogenic agents

The Bioelectrical Properties of Pancreatic Islet Cells: Effect of Diabetogenic Agents Diabetologia 0 0 D e p a r t m e n t of Pharmacology, University of Cambridge , Hills Road, Cambridge, England Summary. Alloxan 5 mM depolarised the islet b u t not the aeinar cells of mouse pancreatic segments in vitro. This effect was prevented b y D-glucose b u t not b y glutathione, 3, 0, methyl-e-D-glucose, D-glucosamine, D-mannoheptulose, or L-leucine. P r e t r e a t m e n t of islet /?-cells with streptozotocin 20 mM caused no depolarization b u t inhibited the generation of action potentials b y D-glucose, L-leueine, D-mannose and D-glyceraldehyde, whereas tolbutamide-induced action potentials were not blocked ; the alkylating moiety of streptozotocin, N-nitroso N- m e t h y l urea produced similar effects. Prior exposure of the islet cells to nicotinamide 4.1 mM conferred protection against streptozotoein action. These observations are discussed in relation to the diabetogenic action of alloxan and streptozotocin. Alloxan; D-glucose protection; islet cell potentials; D-mannoheptulose; mouse pancreas; nieotiaamide protection; streptozo~ocin; N-nitroso; N-methyl Propridt~s biodlectriques des cellules des ~lots pancrdatiques: Effets des agents diabdtog~nes Rgsumd. L'alloxane (5 raM) a d6polarisd les cellules des ilots, mais non les cellules de l'acinus des segments pancrdatiques chez la souris in vitro. Cet effet 6tait anihil6 par le D-glucose, mais non p a r le glutathion, le 3, 0, mdthyl-~-D-glueose, la D-glucosamine, le D-mannoheptulose ou la L-leucine. Le pr6-traitement des eellules fl des ilots ~ la streptozotoeine (20 mM) n ' a pas provoqu6 de d6polarisation, mais a inhibd la gdngration de potentiels d'action par le D-glucose, la L'leueine, le D-mannose et la D-glyc6ralddhyde, tandis que les potentiels d'action provoqu~s par la t o l b u t a m i d e n'dtaient pus bloquds; la partie a l k y l a n t e de la strep~ozotoeine, la N-nitroso-Nm g t h y l ur6e produisaient les mgmes effets. L'exposition prdalable des cellules des ilots ~ l'amide nicotinique (4.1 mM) fournissait une protection contre l'aetion de la streptozotoeine. Ces observations sont diseut~es en relation avee Faction diabdtoggne de l'Mloxane et de la streptozotocine. Introduction The d i a b e t i c s t a t e in an a n i m a l can be i n d u c e d p o t e n t i a l can be affected b y e x p o s u r e to glucose, mannose, leueine a n d t o l b u t a m i d e . These substances, which are also k n o w n to evoke insulin secretion, induce p e r m a n e n t l y b y t h e selective c y t o t o x i c a g e n t s Mloxan small a c t i o n p o t e n t i a l s in t h e islet cells (Dean and a n d s t r e p t o z o t o e i n . H o w e v e r , t h e m e t a b o l i c a b n o r m a l M a t t h e w s , 1970a; M a t t h e w s a n d Dean, 1970) . Changes ities of d i a b e t e s m e l l i t u s i n d u c e d b y these c o m p o u n d s in t h e ionic c o m p o s i t i o n of the e x t r a c e l l u l u r fluid can are n o t identical, suggesting t h a t t h e y h a v e a different influence t h e electrical c h a r a c t e r i s t i c s of t h e a c t i o n m e c h a n i s m of a c t i o n ( N a n s f o r d a n d Opie, 1968) . Allop o t e n t i a l s (Dean a n d M a t t h e w s , 1970b) . I n this p a p e r x a n is speeificMly t a k e n u p b y mouse islet tissue a n d t h e effects of Mloxan a n d s t r e p t o z o t o c i n on mouse paneytologieM changes are visible in fi-eells w i t h i n 5 m i n ereatic islet cell m e m b r a n e p o t e n t i a l s a n d on electrical of Mloxan a d m i n i s t r a t i o n (I-Iammarstrom, t I e l l m a n a c t i v i t y are described. A p a r a l l e l s t u d y has been m a d e a n d Ullberg, 1966; L a z a r u s , B u r d e n a n d B r a d s h a w , of t h e a c t i o n of t h e c o m p o u n d N - n i t r o s o N - m e t h y l 1962) . The r a p i d increase in the r a t e of p e n e t r a t i o n of urea, t h e M k y l a t i n g m o i e t y of s t r e p t o z o t o c i n , on t h e l t C - m a n n i t o l i n t o t o a d f i s h islets i n d i c a t e d t h a t Mloxan electrical a c t i v i t y i n d u c e d b y glucose in islet cells. a l t e r e d t h e p e r m e a b i l i t y of /~-eells ( W a t k i n s , Cooperstein a n d L a z a r o w , 1964) . L i t t l e is k n o w n a b o u t t h e m e c h a n i s m of i n d u c t i o n of d i a b e t e s b y s t r e p t o z o t o c i n . E l e e t r o p h y s i o l o g i c a l studies h a v e shown t h a t fiThe p a n c r e a s was r e m o v e d from albino mice of cells from mouse p a n c r e a t i c islets h a v e a cellular t r a n s m e m b r a n e p o t e n t i a l of --20.1 m V a n d t h e resting either sex weighing 3 0 - - 4 0 g. A s e g m e n t of p a n c r e a s was p l a c e d in a p e r s p e x tissue b a t h a n d superfused Methods P.M. Dean and E . K . Matthews: The Bioelectrical Properties of Pancreatic Islet Cells 5ram AIIoxan +1 0 10 20 30 Z,O 50 60 min. Fig. 1. The effect of alloxan on the membrane potential of islet cells from mouse pancreas. The first eolum shows the mean membrane potential obtained during the 60 min period before addition of alloxan t r a n s m e m b r a n e potentials according to the methods described previously (Dean and Matthews, 1970a) . Since alloxan, streptozotocin and N-nitroso Nm e t h y l urea are rapidly decomposed b y hydrolysis, solutions of these compounds were made immediately before use and added directly to the tissue bath. The possible methylation of nieotinamide b y streptozotocin was investigated chromatographically. 20 mM streptozotocin and 4.1 mM nicotinamide solutions were incubated at 37~ for 20 rain and a 100 ~L aliquot applied to W h a t m a n No. 1 filter paper. Aliquots (100 ~L) of solutions of nicotinamide 4.1 raM, N-methylnicotinamide 4.1 mM and 1 m e t h y l nieotinamide 4.1 mM were used for comparison. The chromategrams were developed in N-butanol saturated with water, and the spots visualized after exposure to iodine vapour. Results Experiments with alloxan The effect of the addition of alloxan 5 m ~ on mouse islet cell m e m b r a n e potentials is shown in Fig. 1. Alloxan did not affect the acinar cell m e m b r a n e potentials, whereas the islet cells showed a rapid, significant, and sustained depolarization from --20.8 m V  S.E.M. 2.4 m V to - - 8 . 6 m V 4 - S . E . M . 1.3 m V ( P < 0 . 0 1 ) . ~ T ;mV --+-j_ 4 J~ ~-20-15-5 -~ G SH GLUCOSE L-LEUCINE MANNOHEPTULOSE 3"O'METHYL D-GLUCOSAMINE 15mM 16.6mM 15 m M 23 .SmM 25"G6LmUMCOSE 23'2 mM Fig. 2. The histograms show the effectof preineubation of isletcellsfor 15 rain with various compounds, indicated beneath each histogram. The Ist column shows the m e a n m e m b r a n e potential in normal solution,the 2nd column shows the effect of alloxan 5 m M after preincubation with a possible protective compound, the m e m b r a n e potentials being measured during the 30-- 60 min period after alloxan. The inset oscilloscopetraces show electricalactivity during the preincubation period with glucose 16.6 mM and L-leueine 15 mM with Krebs-Henseleit solution at 37~ The solution had the following composition: NaC1 103 raM, KC1 4.7 raM, CaCls 2.56 raM, MgC12 1.13 raM, NaHCOs 25 raM, NaHePO4 1.15 mM, D-glucose 2.8 raM, sodium p y r u v a t e 4.9 raM, sodium f u m a r a t e 2.7 raM, sodium glutamate 4.9 raM. I t was gassed with 95% 0 2 - - 5 % COs. The islets of Langerhans were separated from the surrounding acinar tissue b y micro-dissection. Glass micro-electrodes filled with 1.5 M potassium citrate, tip resistance 100 M f~, were used to record the cellular Alloxan did not induce electrical activity in islet cells. Histograms of frequency plotted against m e m b r a n e potential for impalements obtained during the period 30 to 60 rain after alloxan 5 mM did not reveal a population of islet cells which were resistant to depolarization b y alloxan. The results shown in Fig. 2 are the effects of preincubation of islets with glutathione 15 mM, I)-glueose 16.6 mM, L-leucine 15 mM, 3,0-methyl-c~-D-glucose 25.6 mM, D-glueosamine 23.2 raM, or D-mannoheptu P.M. Dean and E.K. Matthews: The Bioeleetrical Properties of Pancreatic Islet Cells lose 23.8 mlV[, on the depolarization of islet cells normally produced b y alloxan 5 raM. The mean m e m b r a n e potential was measured for impalements obtained during the control period (60 min) before exposure to alloxan (this value is shown on the left column of each histogram). Preincubation with 15 mM g]utathione 15 rain before and during exposure to alloxan 5 mM did not prevent depolarization (values shown in the right-hand column of each histogram were obtained from the period 30--60 min after alloxan administration). I n contrast, glucose 16.6 mM, produced complete protection against alloxan. During the superfusion with glucose 16.6 mM action potentials were generated in islet cells (Fig. 2). Preincubation with Lleueine 15 mM, which also induced action potentials, failed to protect against the depolarization produced b y alloxan 5 raM. The strnetural analogues of glucose : 3,0-methyl-~-D-glueose, D-glueosamine and mannoheptulose -- an inhibitor of hexokinase, failed to prevent the depolarization of islet cells. These drugs when exposed to the tissue alone, did not produce depolarization. Experiments with streptozotocin Streptozotoein is rapidly inactivated b y hydrolysis at physiological p I I (Garret, 1960) so t h a t the drug could not be superfused over islet cells for more t h a n short time periods. The islet tissue was perfused for one hour in normal Krebs-Henseleit solution and numerous islet cells were impaled in order to determine a mean value for the m e m b r a n e potential. A dose of steptozotocin was added directly to the b a t h to achieve an initial concentration of 20 mM, the m e m b r a n e potentials were measured, and the mean values obtained for successive 10 rain intervals. During the 60 rain period after exposure to streptozotocin the m e m b r a n e potential was unchanged (Fig. 3) and streptozotoein did not itself induce electrical activity in islet cells. Elsewhere it has been shown t h a t D-glucose 11.1 raM, L-leucine 10 raM, D-mannose 16.6 mM and Dglyceraldehyde 11 mM each induce electrical activity in islet cells (Dean and Matthews, 1970a; Matthews and Dean, 1970; Dean and Matthews, 1971) . This t y p e of activity was characterized b y bursts of action potentials occurring at 10 s intervals; the action potentials had an amplitude of 1 - - 4 mV and a duration of 50 mS. Tolbutamide 0.7 mM, however, induced continuous firing of longer duration action potentials at a rate of a b o u t 1 per sec, 250 ms duration and amplitude 1 - - 8 mV. The effect of streptozotocin on action potential generation was studied as follows. Measurements of islet cell m e m b r a n e potentials were made during a 60 min control period in normal Krebs-I-Ienseleit solution containing the stimulating agent, e.g. D-glucose 11.1 mN[ and the fraction of impaled cells which produced electrical activity was noted; this procedure was repeated on several islet preparations. I n further experiments, m e m b r a n e potentials were measured during a control period, then, after a single dose of streptozototin corresponding to a peak b a t h concentration of 20 mM, m e m b r a n e potentials were measured during the next 60 min; the preparation was subsequently superfused for 30 rain with glucose 11.1 mM (or other stimulating agent) and the fraction of cells exhibiting action potentials during this 30 rain period was observed. The results expressed in Table 1 show t h a t p r e t r e a t m e n t with streptozotoein inhibited the generation of action potentials b y D-glucose, L-leucine, D-mannose and D-glyceraldehyde, whereas tolbutamide-induced action potentials were not blocked b y p r e t r e a t m e n t with streptozotoein. 20ram STREPTOZOTOC[N 252015105mV Nicotinamide injected intravenously into mice protects against the diabetogenic properties of streptozotocin (Schein and Bates, 1968) . After obtaining control values of m e m b r a n e potentials in normal KrebsHenseleit solution, the cells were exposed to solution containing nieotinamide 4.1 mM 15 rain before addition of streptozotocin 20 raM; 60 min later solution containing glucose 11.1 mM was perfnsed over the tissue for 30 rain. Glucose induced firing in 83% of the impaled islet cells showing t h a t nicotinamide conferred protection against streptozotocin. Exposure to nieotinamide 4.1 mM alone did not generate action potentials or change the resting m e m b r a n e potential. The effect of N-nitroso N - m e t h y l urea was investigated for comparison with streptozotocin. Membrane potentials were measured in normal Krebs-Henseleit solution during a 60 min control period, then N-nitroso N - m e t h y l urea was added to the b a t h to produce a Compound inducing action potentials concentration D-glucose 11.1 mM D-mannose 16.6 mM L-leucine 10 mM D-glyceraldehyde 11 mM Tolbutamide 7 10-4M nicotinamide pretreatment, } followed by D- 11.1 mM glucose Compound inducing action potentials concentration D-glucose Tolbutamide ll.1 mM 7 1O-4M u = number of experiments. P.M. Dean and E.K. Matthews : The Bioelectrical Properties of Pancreatic Islet Cells peak concentration of 20 mlYi and the preparation was superfused for 60 min. When the tissue was exposed to a solution containing D-glucose 11.1 raM, action potentials were induced in only 3.5% of the cells. On the other hand with tolbutamide 0.7 m N as a stimulating agent, action potentials with characteristics typical of tolbutamide were produced in 50% of the cells. (Table 1). Paper chromatography showed t h a t incubation of streptozotocin 20 m N with nicotinamide 4.1 mM produced a single spot with an RF value of 0.65; nicotinamide itself had an t~r value of 0.66. No spots were found corresponding to N-methyl nicotinamide (RF accompanying changes in islet cell permeability to ~4Cmannitol (Watkins, Cooperstein and Lazarow, 1964) . In contrast, streptozotocin did not alter islet cell membrane potentials during the 90 min period of exposure; the earliest cytological ehanges are apparently observed only after 2 h (Brosky and Logothetopoulos, 1969) . Of the substances tested, only D-glucose protected islet cells against depolarization b y alloxan. There is no evidence for inactivation of alloxan b y glucose (Webb, 1966) . Since D-glucose, D-fructose and Dmannose given in vivo protect against alloxan diabetes (Bhattaeharya, 1953; 1954) and all three hexoses are 0.82) or 1-methyl nieotinamide (RF 0.05). Therefore, the possibility of simple chemical inactivation of streptozotoein, with nicotinamide acting as an acceptor for the free methyl radical, seems unlikely. Discussion Alloxan rapidly and selectively depolarized the cells from mouse islets of Langerhans. In contrast, guinea-pig islet cells, which are known to be resistant to the diabetogenic properties of alloxan (Maske and Weinges, 1957) , are not depolarized by Mloxan (Dean and Matthews, 1968) . The rate of onset of depolarization b y alloxan, an effect occurring within l0 min, correlates well with the time course of cytological changes which are: a diminution in nuclear and cytoplasmic granules, vacuolation and shrinkage of cytoplasm (Lazarus, Barden and Bradshaw, 1962), with metabolized b y hexokinase, it was thought t h a t alloxan might act b y inhibiting hexokinase. However, VillarPalasi, Carbadillo, Sols and Arteta (1957) showed t h a t the ratio of the protective properties of D-glucose, Dmannose and D-fructose did not correspond to the affinity of these hexoses for pancreatic hexokinase. The glucose analogues 3,0-methyl-~-D-glueose and D-glueosamine did not confer protection, neither did D-mannoheptulose which is a specific inhibitor of hexokinase. Since glucose induced action potentials in islets, the possibility occurred t h a t some property related to the action potential discharge could have produced the protection. However, this is improbable because L-leucine, which also induced action potentials, did not protect the islet cells from the depolarizing influence of alloxan. Although glutathione given in vivo protects against alloxan diabetes (Lazarow, 1946) , no in vitro protection was observed. ~o of cells induced 0/0 of cells induced n to fire action po- to fire action potentials by the tentials by the compound without compound after pretreatment with pretreatment with streptozotoein streptozotoein 78~o 84~o 53% 67% 70~o 78% 00/0 0~ 0% 0% 67.5~ 83~ % of cells induced % of cells induced n to fire action po- to fire action potentials by the tentials by the compound without compound after pretreatment with pretreatment with N-nitroso N-nitroso N-methyl urea N-methyl urea 78% 70~ 3.5% 50~ 4 4 7 5 6 7 6 P.M. Dean and E.K. Matthews: The Bioelectrieal Properties of Pancreatic Islet Cells P r e t r e a t m e n t of islet cells with streptozotoein destroyed the ability of the cells to generate action potentials when stimulated b y D-glucose, D-mannose, L-leucine or D-glyceraldehyde. I t has recently been shown t h a t preincubation of isolated islets of Langerhans with streptozotoein inhibits glucose-induced insulin secretion (Golden, Baird, Malaisse, Malaisse-Lagae and Walker, 1971) . Induction of electrical activity with tolbutamide was not inhibited b y streptozotocin. I n vivo experiments with mice pretreated with streptozotocin have shown t h a t tolbutamide reduces the diabetic hyperglycemia, p r o b a b l y b y releasing insulin ( g e r u p and Tarding, 1969) . Streptozotocin can therefore distinguish between the two ionic mechanisms in the fl-cell m e m b r a n e b y inhibiting fast action potentials characteristic of D-glucose, L-leueine, D-mannose and D-glyeeraldehyde, whilst not m a r k e d l y affecting the slower action potentials produced b y tolbutamide. P r e t r e a t m e n t with nicotinamide protects isolated r a t islets of Langerhans, and the whole animal, against the diabetogenic action of streptozotoein (Schein and Bates, 1968 ; Sehein, Cooney and Vernon, 1967 ; Golden, Baird, Malaisse, Malaisse-Lagae and Walker, 1971) . Streptozotoein produces a depression of N A D and NADI-I levels in mouse liver; this depression is prevented b y nieotinamide administered before the dose of streptozotoein (Schein and Loftus, 1968) . I t is not known, however, whether streptozotoein produces diabetes b y affecting N A D levels in fi-eells, although our results and those of Golden et al. (1971) suggest this possibility. N-Nitroso N - m e t h y l urea is alleged to be nondiabetogenic (Sehein and Loftus, 1968) . However, in vitro a single dose of N-nitroso N - m e t h y l urea had a similar effect to streptozotoein b y inhibiting action potentials when islet cells were subsequently exposed to glucose. Similarly N-nitroso N - m e t h y l urea did not inhibit action potentials generated b y tolbutamide. The close resemblance between the effects of N-nitroso N - m e t h y l urea and streptozotoein might suggest a similar mode of action on islet cells. The p r i m a r y target for the cellular effect of streptozotoein is unknown; specific inhibition of the glueoreceptor appears unlikely since action potentials elicited with the structurally dissimilar substances, L-leucine and D-glyeeraldehyde are also blocked. The mechanism of insulin secretion is not necessarily destroyed since the hypoglyeemie effect of tolbutamide and action potential genesis still occur after pretreatm e n t with streptozotoein. As the effect of streptozotocin on electrical activity can be mimicked b y N-nitroso N - m e t h y l urea it is probable t h a t the N-nitroso Nm e t h y l urea moiety of streptozotoein causes diabetes, the glucose m o i e t y acting to confer high specificity for the fl-eell, since these cells m u s t have a very specialized mechanism for reeognising and handling glucose molecules. Magee and Schoental (1964) have suggested t h a t alkyl nitroso compounds react with cell thiol groups with the eventual liberation of a m e t h y l radical. The free m e t h y l radical is then able to m e t h y l a t e a n y free sulphydryl or carboxylie groups. H o w alloxan causes islet Bells to depolarize is not y e t known. Inhibition of energy production as the p r i m a r y effect of alloxan seems improbable because dinitrophenol, which is believed to inhibit the production of A T P in m a m m a l i a n cells, does not depolarize the islet cells (Dean and Matthews, 1970a) . Alloxan is normally transported into islet cells, b u t not in cells obtained from mice resistant to alloxan ( H a m m a r strom, st al., 1966), suggesting t h a t the cytotoxieity of alloxan is dependent on uptake into the islets. Since alloxan causes a rapid permeability change it is possible t h a t it reacts directly with a specific membrane component, possibly through the highly reactive earbonyl grouping at position 5. The binding of glucose to the m e m b r a n e m a y induce a eonformational change in the alloxan carrier mechanism, t h e r e b y preventing alloxan from entering the Bell to exert cytological damage (Sehneyius and T/~ljedal, 1971) . However, the precise molecular mechanism for the production of diabetes by either alloxan or streptozotocin remains to be elucidated. Acknowledgements. This work was supported by a Medical l~esearch Council research grant to E.K.M.P.M. D. is a Research Fellow of King's College, Cambridge. We are grateful to Professor P.N. Magee for the supply of N-nitroso N-methyl urea and the Upjohn Company (Kalamazoo) for the supply of streptozotocin. J ~ f er~nce8 Bhattacharya , G. : Protection against alloxan diabetes by mannose and fructose . Science 117 , 230 -- 231 ( 1953 ). - - O n the protection of alloxan diabetes by hexoses . Science 120 , 841 -- 843 ( 1954 ). Brosky , G. , Logothetopoulos , J. : Streptozotocin diabetes in the mouse and guinea-pig . Diabetes 18 , 606 -- 611 ( 1969 ). Dean , P.M. , Matthews , E . K . : Alloxan on islet cell membrane potentials . Brit. J. Pharmac . 34 , 677 ( 1968 ). -- -- Glucose -induced electrieM activity in pancreatic islet cells . J. Physiol . 210 , 255 -- 264 ( 1970a ). -- - - Electrical activity in pancreatic islet cells: effect of ions . J. Physiol . 210 , 255 -- 275 ( 1970b ). -- - - The effect of some metabolic intermediates on islet cell membrane potentials . Bioehem. biophys. Acta ( 1971 ) in the press. 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Cancer Res . 27 , 2324 -- 2332 ( 1967 ). -- Loftus , S.: Streptozotocin: depression of m o u s e liver pyridine nucleotides . Cancer Res . 28 , 1501 -- 1506 ( 1968 ). Schneyius , A. , T/iljedal, I . B . : On the mechanism of glucose protection against alloxan toxicity . Diabetologia 7 , 252 255 ( 1971 ). Villar-Palasi , C. , Carbadillo , A. , Sols , A. , Arteta , J . L . : Sensitivity of pancreas hexokinase towards alloxan a n d its modification b y glucose . Nature 180 , 387 -- 388 ( 1957 ). Watkins , D. , Cooperstein , S.J. , Lazarow , A. Effect of alloxan on permeability of pancreatic islet tissue in vitro . Amer. J. Physiol . 207 , 436 -- 440 ( 1964 ). Webb , J . L . : Alloxan. I n : E n z y m e a n d metabolic inhibitors . Vol. 3 , p. 390 . New York: Academic Press 1966.

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P. M. Dean, E. K. Matthews. The bioelectrical properties of pancreatic islet cells: Effect of diabetogenic agents, Diabetologia, 1972, 173-178, DOI: 10.1007/BF01212257